Thrombotic thrombocytopenic purpura (“TTP” or Moschcowitz disease) is a severe and rare disorder of the blood-coagulation system, causing extensive microscopic blood clots to form in the small blood vessels throughout the body. Most cases of TTP arise from deficiency or inhibition of the enzyme ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 domains 13). ADAMTS13 is the proteolytic enzyme responsible for cleaving large multimers of von Willebrand factor (VWF) and is also known as VWF cleaving protease. Thus, there is a relationship between the biological function of ADAMTS13 and the existence of ultra-large molecular weight multimers of VWF and the occurrence of TTP or TTP-like clinical symptoms. TTP also may be related to cancer, chemotherapy, HIV infection, hormone replacement therapy and estrogens, and a number of commonly used medications (including ticlopidine, clopidogrel, and cyclosporine A).
A low level of ADAMTS13 causes clotting substances (platelets) in the blood to clump. As the platelets clump together, there are fewer platelets available in the bloodstream. This clumping, or aggregation, can lead to bleeding under the skin and purple-colored spots called purpura. It also can cause red blood cells to break apart (undergo hemolysis) as they are subjected to shear stress as they pass the microscopic platelet clots. Red blood cells are thus destroyed prematurely. Reduced blood flow and cellular injury results in end organ damage. Current therapy is based on support and plasmapheresis to reduce circulating antibodies against ADAMTS13 and replenish blood levels of ADAMTS13.
Development of antibodies to protein therapeutics is a persistent problem when biopharmaceuticals are used for treatment of disorders like TTP and hemophilia. These antibodies often inhibit the activity of the protein therapeutic thereby reducing the efficacy of the treatment or requiring increasing doses of drug to maintain therapeutic levels. Because these blood disorders are often lifelong conditions, the appearance of antibodies specific for therapeutic blood clotting factors is particularly trying for patients receiving the treatment and challenging for doctors treating these patients.
The role that preclinical models play in the evaluation of drug efficacy and optimization of lead compounds is an essential one in pharmaceutical companies. Without a robust, dependable animal model of human disease, the design of better molecules becomes a daunting task. For this objective, transgenic and knockout mouse and rat models have held great promise, but yet have been underutilized in the pharmaceutical industry. The limited use of such models is likely due in part to the failure of many current transgenic and knockout models to exhibit essential qualities of preclinical screening models; validity, reliability, and utility.
In an effort to better understand TTP and the potential for therapy, an animal model for the disorder has been sought. Early attempts to recreate a TTP model relied on chemical induction using, e.g., venom factor botrocetin or 2-butoxyethanol (BE). Botrocetin acts by binding and multimerizing VWF, resulting in platelet aggregation. Animals treated with the factor exhibit transient thrombocytopenia, but not all of the symptoms associated with TTP (Sanders et al., Arterioscler. Thromb. Vasc. Biol. 15:793-800, 2005; Brinkhous et al., Mayo Clin. Proc. 66:733-42, 1991). Similarly, BE-treated animals developed certain symptoms of TTP, including hemolysis and thrombosis (Ezov et al., Cardiovasc. Toxicol. 2:181-93, 2002). However, the model fails to exhibit all of the hallmark symptoms of TTP. Later attempts involved generation of ADAMTS13 deficient mice. In most genetic backgrounds, however, the phenotype is minimal, indicating that ADAMTS13 deficiency is not sufficient to cause TTP (Banno et al., Blood 107:3161-66, 2006; Desch et al., Arterioscler. Thromb. Vasc. Biol. 27:1901-08, 2007).
Currently, no valid animal model is available to test therapies for the treatment of TTP. Therapies are limited, and include such procedures as plasma treatment, plasma exchange, and splenectomy. Thus, there exists a need in the art to develop such a model and to develop methods to study the effects of various TTP therapies in vivo without study on human patients. Further, there remains a need in the art to determine if administration of exogenous therapeutic protein to a patient will result in production of antigen-specific antibodies which inhibit protein activity in vivo.